Project Summary. Selenium, an essential micronutrient for human health, exerts its biological role as the amino acid selenocysteine (Sec). Selenium deficiency, mutations in selenoprotein genes, and low levels of selenoproteins lead to diseases affecting various organ systems. Also, inefficient Sec incorporation diminishes selenoenzyme activity and selenoprotein structure. Despite its significance, the mechanism of Sec incorporation into a nascent selenoprotein is not well understood. This question is at the focus of our original proposal. However, the absence of a method permitting analysis of the composition and stoichiometry of ribonucleoprotein complexes that catalyze specific reactions of Sec formation creates barriers to progress and innovation. We seek funding to obtain multi-angle light scattering and refractive index (MALS-RI) detector that will enable us to precisely characterize multimolecular assemblies typifying anabolic cycle of Sec. According to the current model, the specialized elongation factor, eEFSec, delivers Sec-tRNASec to the ribosome in response to an in-frame UGA codon. Accurate decoding of the UGA codon also requires auxiliary protein (SBP2) and RNA (SECIS) factors that are absent from the canonical translational machinery. Understanding structure, composition, architecture and stoichiometry of macromolecular assemblies governing Sec incorporation will advance our understanding of selenoprotein synthesis. In the original project, we proposed to use size-exclusion chromatography coupled to small-angle X-ray scattering (SEC-SAXS) to analyze both the eEFSec-tRNASec complex (Specific Aim 1) and the ?recruitment? complex comprised of SBP2, SECIS, eEFSec, and Sec-tRNASec (Specific Aim 2). SEC-SAXS is quite powerful but the sample requirements correlate with the target size and, at some point, this method becomes too prohibitive to use. Also, SEC-SAXS is often not sensitive enough to detect oligomers, aggregates and complexes of slightly different compositions that may be present in the sample. Clearly, having access to an approach that would be able to discern among distinct macromolecular species in the same sample would be instrumental to success of our structural and mechanistic studies. Recently, we probed the utility of the MALS-RI detector for our studies. Because of the instrument configuration, individual ribonucleoprotein complexes, proteins and nucleic acids were eluted from the SEC column through the MALS-RI detector and then through the SAXS sample chamber. This particular configuration enabled concurrent collection and comparison of the MALS-RI and SAXS data. After running a series of tests, the conclusion is that MALS-RI should be routinely used in our studies, particularly when multicomponent systems are being studied. However, MALS-RI is not easily accessible within the Chicagoland area. The only detector is at the BioCAT beamline, but this particular detector can only be used when the beam station is not used. This limitation creates obvious barriers to the progress of our research. This prompted us to put forward this proposal with the goal of obtaining funding for the purchase of the MALS-RI detector, which will upgrade the existing HPLC system in the laboratory. This detector will allow simple, rapid, precise and timely analyses of complexes typifying Sec cycle. The instrument features low sample volume requirement, high sensitivity and it can be installed onto one of our existing Shimadzu HPLC instruments. Routine and unlimited access to MALS-RI will greatly facilitate completion of the proposal and other related studies on important questions in the field of selenium biology and selenoprotein gene translation.